This invention relates to novel pharmaceutically useful compounds, in particular compounds that are, and/or compounds that are metabolised to compounds which are, competitive inhibitors of trypsin-like serine proteases, especially thrombin, their use as medicaments, pharmaceutical compositions containing them and synthetic routes to their production.
Blood coagulation is the key process involved in both haemostasis (i.e. the prevention of blood loss from a damaged vessel) and thrombosis (i.e. the formation of a blood clot in a blood vessel, sometimes leading to vessel obstruction).
Coagulation is the result of a complex series of enzymatic reactions. One of the ultimate steps in this series of reactions is the conversion of the proenzyme prothrombin to the active enzyme thrombin.
Thrombin is known to play a central role in coagulation. It activates platelets, leading to platelet aggregation, converts fibrinogen into fibrin monomers, which polymerise spontaneously into fibrin polymers, and activates factor XIII, which in turn crosslinks the polymers to form insoluble fibrin. Furthermore, thrombin activates factor V and factor VIII leading to a xe2x80x9cpositive feedbackxe2x80x9d generation of thrombin from prothrombin.
By inhibiting the aggregation of platelets and the formation and crosslinking of fibrin, effective inhibitors of thrombin would be expected to exhibit antithrombotic activity. In addition, antithrombotic activity would be expected to be enhanced by effective inhibition of the positive feedback mechanism.
The early development of low molecular weight inhibitors of thrombin has been described by Claesson in Blood Coagul. Fibrinol. (1994) 5, 411.
Blombxc3xa4ck et al (in J. Clin. Lab. Invest. 24, suppl. 107, 59, (1969)) reported thrombin inhibitors based on the amino acid sequence situated around the cleavage site for the fibrinogen Axcex1 chain. Of the amino acid sequences discussed, these authors suggested the tripeptide sequence Phe-Val-Arg (P9-P2-P1, hereinafter referred to as the P3-P2-P1 sequence) would be the most effective inhibitor.
Thrombin inhibitors based on dipeptidyl derivatives with an xcex1,xcfx89-aminoalkyl guanidine in the P1-position are known from U.S. Pat. No. 4,346,078 and International Patent Application WO 93/11152. Similar, structurally related, dipeptidyl derivatives have also been reported. For example International Patent Application WO 94/29336 discloses compounds with, for example, aminomethyl benzamidines, cyclic aminoalkyl amidines and cyclic aminoalkyl guanidines in the P1-position (International Patent Application WO 97/23499 discloses prodrugs of certain of these compounds); European Patent Application 0 648 780, discloses compounds with, for example, cyclic aminoalkyl guanidines in the P1-position.
Thrombin inhibitors based on peptidyl derivatives, also having cyclic aminoalkyl guanidines (e.g. either 3- or 4- aminomethyl-1-amidino-piperidine) in the P1-position are known from European Patent Applications 0 468 231, 0 559 046 and 0 641 779.
Thrombin inhibitors based on tripeptidyl derivatives with arginine aldehyde in the P1-position were first disclosed in European Patent Application 0 185 390.
More recently, arginine aldehyde-based peptidyl derivatives, modified in the P3-position, have been reported. For example, International Patent Application WO 93/18060 discloses hydroxy acids, European Patent Application 0 526 877 des-amino acids, and European Patent Application 0 542 525 O-methyl mandelic acids in the P3-position.
Inhibitors of serine proteases (e.g. thrombin) based on electrophilic ketones in the P1-position are also known. For example, European Patent Application 0 195 212 discloses peptidyl xcex1-keto esters and amides, European Patent Application 0 362 002 fluoroalkylamide ketones, European Patent Application 0 364 344 xcex1,xcex2,xcex4-triketocompounds, and European Patent Application 0 530 167 xcex1-alkoxy ketone derivatives of arginine in the P1-position.
Other, structurally different, inhibitors of trypsin-like serine proteases based on C-terminal boronic acid derivatives of arginine and isothiouronium analogues thereof are known from European Patent Application 0 293 881.
More recently, thrombin inhibitors based on peptidyl derivatives have been disclosed in European Patent Application 0 669 317 and International Patent Applications WO 95/35309, WO 95/23609, WO 96/25426, WO 97/02284, WO 97/46577, WO 96/32110, WO 96/31504, WO 96/03374, WO 98/06740, WO 97/49404, WO 98/57932, WO 99/29664 and WO 00/35869. In particular WO 97/02284 and WO 00/42059 disclose thrombin inhibitors with substituted mandelic acids in the P3 position.
However, there remains a need for effective inhibitors of trypsin-like serine proteases, such as thrombin. There is also a need for compounds which have a favourable pharmacokinetic profile (e.g. low clearance) and are selective in inhibiting thrombin over other serine proteases, in particular those involved in haemostatis. Compounds which exhibit competitive inhibitory activity towards thrombin would be expected to be especially useful as anticoagulants and therefore in the therapeutic treatment of thrombosis and related disorders.
According to the invention there is provided compounds of formula I, 
wherein
R1 represents C(O)CH3 or C1-3 alkyl; and
Y represents xe2x80x94CH2xe2x80x94 or xe2x80x94(CH2)2xe2x80x94,
and pharmaceutically-acceptable derivatives thereof.
The term xe2x80x9cpharmaceutically-acceptable derivativesxe2x80x9d includes inter alia pharmaceutically-acceptable salts (e.g. acid addition salts).
Preferred compounds of formula I include those in which:
R1 represents C(O)CH3, methyl or ethyl;
Y represents xe2x80x94CH2xe2x80x94.
Particularly preferred compounds of formula I include
Ph(3-Cl)(5-NHMe)xe2x80x94CH(OH)C(O)-Aze-Pab;
Ph(3-Cl)(5-NHAc)xe2x80x94CH(OH)C(O)-Aze-Pab.
Abbreviations are listed at the end of this specification.
Compounds of formula I may be made in accordance with techniques well known to those skilled in the art, for example as described hereinafter.
According to a further aspect of the invention there is provided a process for the preparation of a compound of formula I, which comprises:
(i) the coupling of a compound of formula II 
wherein R1 is as hereinbefore defined, with a compound of formula III, 
wherein Y is as hereinbefore defined, for example in the presence of a coupling agent (e.g. oxalyl chloride in DMF, EDC, DCC, HBTU, HATU, PyBOP or TBTU), an appropriate base (e.g. pyridine, DMAP, TEA, 2,4,6-collidine or DIPEA) and a suitable organic solvent (e.g. dichloromethane, acetonitrile, EtOAc or DMF);
(ii) the coupling of a compound of formula IV, 
wherein R1 and Y are as hereinbefore defined, with para-amidinobenzylamine, for example under conditions as described in step (i) above; or
(iii) deprotection of a protected derivative of a compound of formula I under standard conditions.
Compounds of formula I may be prepared by way of deprotection of a corresponding compound of formula XV, as defined hereinafter, which deprotection comprises removal of the group C(O)ORX, in which RX is as defined hereinafter, from the compound of formula XV, for example under conditions known to those skilled in the art (e.g. by reacting with QF or TFA (e.g. as described hereinafter)).
Further, compounds of formula I may be prepared by way of deprotection of a corresponding compound of formula Ia, as defined hereinafter, in which R2 represents OR3, wherein R2 and R3 are as defined hereinafter, for example by hydrogenation in the presence of a suitable catalyst (e.g. a supported metal catalyst such as Pd/C (e.g. 10% (w/w) Pd/C)) and an appropriate solvent (e.g. a lower (e.g. C1-6) alkyl alcohol such as ethanol), and optionally in the presence of a suitable acid (e.g. acetic acid).
Compounds of formula II are available using known and/or standard techniques.
For example, compounds of formula II may be prepared by reaction of an aldehyde of formula V, 
wherein R1 is as hereinbefore defined with:
(a) a compound of formula VI,
Rxe2x80x3CN xe2x80x83xe2x80x83VI 
wherein Rxe2x80x3 represents H or (CH3)3Si, for example at room, or elevated, temperature (e.g. below 100xc2x0 C.) in the presence of a suitable organic solvent (e.g. chloroform or methylene chloride) and, if necessary, in the presence of a suitable base (e.g. TEA) and/or a suitable catalyst system (e.g. benzylammonium chloride or zinc iodide), followed by hydrolysis under conditions that are well known to those skilled in the art (e.g. as described hereinafter);
(b) NaCN or KCN, for example in the presence of NaHSO3 and water, followed by hydrolysis;
(c) chloroform, for example at elevated temperature (e.g. above room temperature but below 100xc2x0 C.) in the presence of a suitable organic solvent (e.g. chloroform) and, if necessary, in the presence of a suitable catalyst system (e.g. benzylammonium chloride), followed by hydrolysis;
(d) a compound of formula VII, 
wherein M represents Mg or Li, followed by oxidative cleavage (e.g. ozonolysis or osmium or ruthenium catalysed) under conditions which are well known to those skilled in the art; or
(e) tris(methylthio)methane under conditions which are well known to those skilled in the art, followed by hydrolysis in the presence of e.g. HgO and HBF4.
Compounds of formula II may alternatively be prepared from Ph(3-Cl)(5-NH2)xe2x80x94CH(OH)C(O)OH, for example as described hereinafter for compounds of formula II in which R1 represents C(O)CH3 or methyl.
The enantiomeric forms of the compound of formula II (i.e. those compounds having different configurations of substituents about the C-atom xcex1- to the CO2H group) may be separated by an enantiospecific derivatisation step. This may be achieved, for example by an enzymatic process. Such enzymatic processes include, for example, transesterification of the xcex1-OH group at between room and reflux temperature (e.g. at between 45 and 65xc2x0 C.) in the presence of a suitable enzyme (e.g. Lipase PS Amano), an appropriate ester (e.g. vinyl acetate) and a suitable solvent (e.g. methyl tert-butyl ether). The derivatised isomer may then be separated from the unreacted isomer by conventional separation techniques (e.g. chromatography).
Groups added to compounds of formula II in such a derivatisation step may be removed either before any further reactions or at any later stage in the synthesis of compounds of formula I. The additional groups may be removed using conventional techniques (e.g. for esters of the xcex1-OH group, hydrolysis under conditions known to those skilled in the art (e.g. at between room and reflux temperature in the presence of a suitable base (e.g. NaOH) and an appropriate solvent (e.g. MeOH, water or mixtures thereof))).
Compounds of formula IV may be prepared by coupling a compound of formula II as hereinbefore defined to a compound of formula VIII, 
wherein Y is as hereinbefore defined, for example under similar conditions to those described herein for preparation of compounds of formula I.
Compounds of formula V are available using known and/or standard techniques. For example, they may be prepared by:
(i) reduction of a compound of formula X, 
wherein R1 is as hereinbefore defined, or a protected derivative thereof, in the presence of a suitable reducing agent (e.g. DIBAL-H); or
(ii) oxidation of a compound of formula XI, 
wherein R1 is as hereinbefore defined, or a protected derivative thereof, in the presence of a suitable oxidising agent (e.g. MnO2, pyridinium chlorochromate or a combination of DMSO and oxalyl chloride).
Compounds of formulae III, VI, VII, VIII, X and XI are either commercially available, are known in the literature, or may be obtained either by analogy with the processes described herein, or by conventional synthetic procedures, in accordance with standard techniques, from readily available starting materials using appropriate reagents and reaction conditions (e.g. as described hereinafter).
Compounds of formula I may be isolated from their reaction mixtures using conventional techniques.
In accordance with the present invention, pharmaceutically acceptable derivatives of compounds of formula I also include xe2x80x9cprotectedxe2x80x9d derivatives, and/or compounds that act as prodrugs, of compounds of formula I.
Compounds that may act as prodrugs of compounds of formula I that may be mentioned include compounds of formula Ia, 
wherein R2 represents OR3 or C(O)OR4;
R3 represents H, C1-10 alkyl, C1-3 alkylaryl or C1-3 alkyloxyaryl (the alkyl parts of which latter two groups are optionally interrupted by one or more oxygen atoms, and the aryl parts of which latter two groups are optionally substituted by one or more substituents selected from halo, phenyl, methyl or methoxy, which latter three groups are also optionally substituted by one or more halo substituents);
R4 represents C1-10 alkyl (which latter group is optionally interrupted by one or more oxygen atoms), or C1-3 alkylaryl or C1-3 alkyloxyaryl (the alkyl parts of which latter two groups are optionally interrupted by one or more oxygen atoms, and the aryl parts of which latter two groups are optionally substituted by one or more substituents selected from halo, phenyl, methyl or methoxy, which latter three groups are also optionally substituted by one or more halo substituents); and
R1 and Y are as hereinbefore defined,
and pharmaceutically-acceptable derivatives thereof.
The term xe2x80x9cpharmaceutically-acceptable derivativesxe2x80x9d of compounds of formula Ia includes pharmaceutically-acceptable salts (e.g. acid addition salts).
Alkyloxyaryl groups that R3 and R4 may represent comprise an alkyl and an aryl group linked by way of an oxygen atom. Alkylaryl and alkyloxyaryl groups are linked to the rest of the molecule via the alkyl part of those groups, which alkyl parts may (if there is a sufficient number (i.e. three) of carbon atoms) be branched-chain. The aryl parts of alkylaryl and alkyloxyaryl groups which R3 and R4 may represent include carbocyclic and heterocyclic aromatic (heteroaryl) groups, such as phenyl, naphthyl, pyridinyl, oxazolyl, isoxazolyl, thiadiazolyl (e.g. 1,2,3-thiadiazolyl), indolyl and benzofuranyl and the like.
Alkyl groups which R3 and R4 may represent may be straight-chain or, when there is a sufficient number (i.e. a minimum of three) of carbon atoms, be branched-chain and/or cyclic. Further, when there is a sufficient number (i.e. a minimum of four) of carbon atoms, such alkyl groups may also be part cyclic/acyclic. Such alkyl groups may also be saturated or, when there is a sufficient number (i.e. a minimum of two) of carbon atoms, be unsaturated.
Halo groups with which R3 and R4 may be substituted include fluoro, chloro, bromo and iodo.
When R2 represents C(O)OR4, preferred R4 groups include:
(a) linear, branched or cyclic C3-6 alkyl, for example C4-6 cycloalkyl;
(b) C1-2 alkylaryl groups, such as benzyl, optionally substituted as indicated hereinbefore.
Preferred compounds of formula Ia include those in which R2 represents OR3.
When R2 represents OR3, preferred R3 groups include:
(a) H;
(b) unsubstituted, linear, branched or cyclic C1-8 (e.g. C1-6) alkyl, such as linear C1-3 alkyl (e.g. methyl, ethyl or i-propyl), branched C3-8 alkyl (e.g. i-butyl) or cyclic C4-7 alkyl (e.g. cyclobutyl or cyclohexyl);
(c) C1-3 alkyloxyphenyl (e.g. C2 alkyloxyphenyl), the phenyl group of which is optionally substituted by one or more substituents as indicated hereinbefore (e.g. trifluoromethyl);
(d) C1-2 alkylaryl (e.g. methylaryl), wherein the aryl group is phenyl, pyridinyl, isoxazolyl or thiadiazolyl, which latter four groups are optionally substituted by one or more substituents as indicated hereinbefore (e.g. methoxy, methyl, bromo and/or chloro).
Preferred compounds of formula Ia include those in which R2 represents OR3 and R3 represents:
(i) linear or cyclic (as appropriate), C1-6 (e.g. C1-4) alkyl, such as methyl, ethyl, i-propyl or cyclohexyl; or
(ii) methylaryl, wherein the aryl group is phenyl or isoxazolyl, which latter two groups are optionally substituted in the aryl part by one substituent selected from methoxy, methyl and bromo (e.g. 4-methylbenzyl, 3-methoxybenzyl, 2-bromobenzyl or 5-methyl-3-isoxazolyl).
Compounds of formula Ia may be prepared by one or more of the following methods:
(a) the coupling of a compound of formula II as hereinbefore defined with a compound of formula XII, 
wherein Y and R2 are as hereinbefore defined, for example under similar conditions to those described hereinbefore for synthesis of compounds of formula I;
(b) the coupling of a compound of formula IV, as hereinbefore defined, with a compound of formula XIII, 
wherein R2 is as hereinbefore defined, for example under similar conditions to those described hereinbefore for synthesis compounds of formula I;
(c) for compounds of formula Ia in which R2 represents OH, reaction of a corresponding compound of formula XIV, 
wherein R1 and Y are as hereinbefore defined, with hydroxylamine, for example under conditions known to those skilled in the art;
(d) for compounds of formula Ia in which R2 represents OR3, reaction of a protected derivative of a corresponding compound of formula I which is, for example, a compound of formula XV, 
wherein RX represents, for example, xe2x80x94CH2CH2xe2x80x94Si(CH3)3 or benzyl, and R1 and Y are as hereinbefore defined, or a tautomer thereof, with a compound of formula XVI,
R3ONH2 xe2x80x83xe2x80x83XVI 
wherein R3 is as hereinbefore defined, or an acid addition salt thereof, for example at between room and reflux temperature in the presence of an appropriate organic solvent (e.g. THF, CH3CN, DMF or DMSO), followed by removal of the xe2x80x94C(O)ORX group under conditions known to those skilled in the art (e.g. by reacting with QF or TFA (e.g. as described hereinafter));
(e) for compounds of formula Ia in which R2 represents COOR4, reaction of a corresponding compound of formula I, as hereinbefore defined, with a compound of formula XVII,
L1COOR4 xe2x80x83xe2x80x83XVII 
wherein L1 represents a suitable leaving group, such as halo, and R4 is as hereinbefore defined, for example at or around room temperature in the presence of suitable base (e.g. NaOH, for example in aqueous solution) and an appropriate organic solvent (e.g. methylene chloride); or
(f) for compounds of formula Ia in which R2 represents OCH3 or OCH2CH3, reaction of a corresponding compound of formula Ia in which R2 represents OH with dimethylsulfate or diethylsulfate, respectively, for example in the presence of a suitable base (e.g. an alkali metal hydroxide such as KOH (for example in aqueous solution at e.g. 50 wt. %)) and an appropriate catalyst (e.g. a quaternary ammonium halide such as benzyltrimethylammonium chloride (for example in CH2Cl2 or THF solution at e.g. 10 wt. %)).
Compounds of formula XIV and XV may be prepared by the coupling of a corresponding compound of formula II to, respectively, a compound of formula XVIII, 
wherein Y is as hereinbefore defined, or a compound of formula XIX, 
wherein Y and RX are as hereinbefore defined, for example in each case under similar conditions to those described hereinbefore for synthesis compounds of formula I.
Compounds of formula XIV and XV may alternatively be prepared by coupling of a corresponding compound of formula IV to, respectively, para-cyanobenzylamine, or a compound of formula XX, 
wherein RX is as hereinbefore defined, for example in each case under similar conditions to those described hereinbefore for synthesis compounds of formula I.
Compounds of formula XV may alternatively be prepared by reaction of a corresponding compound of formula XIV with hydroxylamine under conditions known to those skilled in the art, followed by:
(i) reduction of the resulting hydroxyamidine under conditions known to those skilled in the art (e.g. by catalytic hydrogenation); and then
(ii) reaction of the resulting compound of formula I with a compound corresponding to a compound of formula XVII in which, in place of R4, the group RX is present, in which RX is as hereinbefore defined, for example under conditions described above in respect of the preparation of compounds of formula Ia.
Compounds of formulae XII, XVIII and XIX may be prepared by the coupling of a corresponding compound of formula VIII, as hereinbefore defined, to, respectively, a compound of formula XIII as hereinbefore defined, para-cyanobenzylamine, or a compound of formula XX as hereinbefore defined, for example in each case under similar conditions to those described hereinbefore for synthesis of compounds of formula I.
Compounds of formulae XIII, XVI, XVII and XX are either commercially available, are known in the literature, or may be obtained either by analogy with the processes described herein, or by conventional synthetic procedures, in accordance with standard techniques, from readily available starting materials using appropriate reagents and reaction conditions (e.g. as described hereinafter).
Compounds of formula Ia may be isolated from their reaction mixtures using conventional techniques.
Compounds of formula I and Ia, as defined above, and derivatives of either, are referred to hereinafter as xe2x80x9cthe compounds of the inventionxe2x80x9d.
The compounds of the invention may exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention. Particular tautomeric forms that may be mentioned include those connected with the position of the double bond in the amidine functionality in a compound of formula Ia, and the position of the substituent R2.
Compounds of the invention also contain two or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the invention.
Compounds of the invention in which the 
fragment is in the S-configuration are preferred.
Compounds of the invention in which the 
fragment is in the R-configuration are preferred.
The wavy lines on the bonds in the above fragments signify the bond positions of the fragments.
Thus, particularly preferred compounds of the invention include
Ph(3-Cl)(5-NHMe)-(R)CH(OH)C(O)-(S)Aze-Pab; and
Ph(3-Cl)(5-NHAc)-(R)CH(OH)C(O)-(S)Aze-Pab.
It will be appreciated by those skilled in the art that in the processes described above and hereinafter the functional groups of intermediate compounds may need to be protected by protecting groups.
Functional groups that it is desirable to protect include hydroxy, amino and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl groups (e.g. t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl) and tetrahydropyranyl. Suitable protecting groups for carboxylic acid include C1-6 alkyl or benzyl esters. Suitable protecting groups for amino and amidino include t-butyloxycarbonyl, benzyloxycarbonyl or 2-trimethylsilylethoxycarbonyl (Teoc). Amidino nitrogens may also be protected by hydroxy or alkoxy groups, and may be either mono- or diprotected.
The protection and deprotection of functional groups may take place before or after coupling, or before or after any other reaction in the abovementioned schemes.
Protecting groups may be removed in accordance with techniques that are well known to those skilled in the art and as described hereinafter.
Persons skilled in the art will appreciate that, in order to obtain compounds of the invention in an alternative, and, on some occasions, more convenient, manner, the individual process steps mentioned hereinbefore may be performed in a different order, and/or the individual reactions may be performed at a different stage in the overall route (i.e. substituents may be added to and/or chemical transformations performed upon, different intermediates to those mentioned hereinbefore in conjunction with a particular reaction). This may negate, or render necessary, the need for protecting groups.
The type of chemistry involved will dictate the need, and type, of protecting groups as well as the sequence for accomplishing the synthesis.
The use of protecting groups is fully described in xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, edited by J W F McOmie, Plenum Press (1973), and xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, 3rd edition, T W Greene and P G M Wutz, Wiley-Interscience (1999).
Protected derivatives of compounds of the invention may be converted chemically to compounds of the invention using standard deprotection techniques (e.g. hydrogenation). The skilled person will also appreciate that certain compounds of formula Ia may also be referred to as being xe2x80x9cprotected derivativesxe2x80x9d of compounds of formula I.
Some of the intermediates referred to hereinbefore are novel.
According to a further aspect of the invention there is thus provided: (a) a compound of formula II as hereinbefore defined or a protected derivative thereof; (b) a compound of formula IV, as hereinbefore defined, or a protected derivative thereof; (c) a compound of formula XIV, as hereinbefore defined, or a protected derivative thereof; and (d) a compound of formula XV, as hereinbefore defined, or a protected derivative thereof.
Preferred compounds of formula II include Ph(3-Cl)(5-NHMe)xe2x80x94CH(OH)C(O)OH and Ph(3-Cl)(5-NHAc)xe2x80x94CH(OH)C(O)OH. Preferred compounds of formula III include Ph(3-Cl)(5-NHMe)xe2x80x94CH(OH)C(O)-Aze-OH and Ph(3-Cl)(5-NHAc)xe2x80x94CH(OH)C(O)-Aze-OH. Preferred compounds of formula XV include Ph(3-Cl)(5-NHMe)xe2x80x94CH(OH)C(O)-Aze-Pab(Teoc) and Ph(3-Cl)(5-NHAc)xe2x80x94CH(OH)C(O)-Aze-Pab(Teoc).
Medical and Pharmaceutical Use
Compounds of the invention may possess pharmacological activity as such. Compounds of the invention that may possess such activity include, but are not limited to, compounds of formula I.
However, other compounds of the invention (including compounds of formula Ia) may not possess such activity, but may be administered parenterally or orally, and may thereafter be metabolised in the body to form compounds that are pharmacologically active (including, but not limited to, corresponding compounds of formula I). Such compounds (which also includes compounds that may possess some pharmacological activity, but that activity is appreciably lower than that of the xe2x80x9cactivexe2x80x9d compounds to which they are metabolised), may therefore be described as xe2x80x9cprodrugsxe2x80x9d of the active compounds.
Thus, the compounds of the invention are useful because they possess pharmacological activity and/or are metabolised in the body following oral or parenteral administration to form compounds which possess pharmacological activity. The compounds of the invention are therefore indicated as pharmaceuticals.
According to a further aspect of the invention there is thus provided the compounds of the invention for use as pharmaceuticals.
In particular, compounds of the invention are potent inhibitors of thrombin either as such and/or (e.g. in the case of prodrugs), are metabolised following administration to form potent inhibitors of thrombin, for example as may be demonstrated in the tests described below. By xe2x80x9cprodrug of a thrombin inhibitorxe2x80x9d, we include compounds that form (i.e. are metabolised to) a thrombin inhibitor, in an experimentally-detectable amount, and within a predetermined time (e.g. about 1 hour), following oral or parenteral administration (see, for example, Test E below) or, alternatively, following incubation in the presence of liver microsomes (see, for example, Test G below).
The compounds of the invention are thus expected to be useful in those conditions where inhibition of thrombin is required, and/or conditions where anticoagulant therapy is indicated, including the following:
The treatment and/or prophylaxis of thrombosis and hypercoagulability in blood and/or tissues of animals including man. It is known that hypercoagulability may lead to thrombo-embolic diseases. Conditions associated with hypercoagulability and thrombo-embolic diseases which may be mentioned include inherited or acquired activated protein C resistance, such as the factor V-mutation (factor V Leiden), and inherited or acquired deficiencies in antithrombin III, protein C, protein S, heparin cofactor II. Other conditions known to be associated with hypercoagulability and thrombo-embolic disease include circulating antiphospholipid antibodies (Lupus anticoagulant), homocysteinemi, heparin induced thrombocytopenia and defects in fibrinolysis, as well as coagulation syndromes (e.g. disseminated intravascular coagulation (DIC)) and vascular injury in general (e.g. due to surgery).
The treatment of conditions where there is an undesirable excess of thrombin without signs of hypercoagulability, for example in neurodegenerative diseases such as Alzheimer""s disease.
Particular disease states which may be mentioned include the therapeutic and/or prophylactic treatment of venous thrombosis (e.g. DVT) and pulmonary embolism, arterial thrombosis (e.g. in myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis), and systemic embolism usually from the atrium during atrial fibrillation or from the left ventricle after transmural myocardial infarction, or caused by congestive heart failure; prophylaxis of re-occlusion (i.e. thrombosis) after thrombolysis, percutaneous trans-luminal angioplasty (PTA) and coronary bypass operations; the prevention of re-thrombosis after microsurgery and vascular surgery in general.
Further indications include the therapeutic and/or prophylactic treatment of disseminated intravascular coagulation caused by bacteria, multiple trauma, intoxication or any other mechanism; anticoagulant treatment when blood is in contact with foreign surfaces in the body such as vascular grafts, vascular stents, vascular catheters mechanical and biological prosthetic valves or any other medical device; and anticoagulant treatment when blood is in contact with medical devices outside the body such as during cardiovascular surgery using a heart-lung machine or in haemodialysis; the therapeutic and/or prophylactic treatment of idiopathic and adult respiratory distress syndrome, pulmonary fibrosis following treatment with radiation or chemotherapy, septic shock, septicemia, inflammatory responses, which include, but are not limited to, edema, acute or chronic atherosclerosis such as coronary arterial disease and the formation of atherosclerotic plaques, cerebral arterial disease, cerebral infarction, cerebral thrombosis, cerebral embolism, peripheral arterial disease, ischaemia, angina (including unstable angina), reperfusion damage, restenosis after percutaneous trans-luminal angioplasty (PTA) and coronary artery bypass surgery.
Compounds of the invention that inhibit trypsin and/or thrombin may also be useful in the treatment of pancreatitis.
The compounds of the invention are thus indicated both in the therapeutic and/or prophylactic treatment of these conditions.
According to a further aspect of the present invention, there is provided a method of treatment of a condition where inhibition of thrombin is required which method comprises administration of a therapeutically effective amount of a compound of the invention to a person suffering from, or susceptible to, such a condition.
The compounds of the invention will normally be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route or via inhalation, in the form of pharmaceutical preparations comprising active compound either as a free base, or a pharmaceutically acceptable non-toxic organic or inorganic acid addition salt, or other derivative, in a pharmaceutically acceptable dosage form.
Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.
The compounds of the invention may also be combined and/or co-administered with any antithrombotic agent with a different mechanism of action, such as the antiplatelet agents acetylsalicylic acid, ticlopidine, clopidogrel, thromboxane receptor and/or synthetase inhibitors, fibrinogen receptor antagonists, prostacyclin mimetics and phosphodiesterase inhibitors and ADP-receptor (P2T) antagonists and inhibitors of carboxypeptidase U (CPU).
The compounds of the invention may further be combined and/or co-administered with thrombolytics such as tissue plasminogen activator (natural, recombinant or modified), streptokinase, urokinase, prourokinase, anisoylated plasminogen-streptokinase activator complex (APSAC), animal salivary gland plasminogen activators, and the like, in the treatment of thrombotic diseases, in particular myocardial infarction.
According to a further aspect of the invention there is thus provided a pharmaceutical formulation including a compound of the invention, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
Suitable daily doses of the compounds of the invention in therapeutic treatment of humans are about 0.001-100 mg/kg body weight at peroral administration and 0.001-50 mg/kg body weight at parenteral administration.
The compounds of the invention have the advantage that they may be more efficacious, be less toxic, be longer acting, have a broader range of activity, be more potent, produce fewer side effects, be more easily absorbed, and/or have a better pharmacokinetic profile (e.g. lower clearance), than, or have other useful pharmacological, physical, or chemical, properties over, compounds known in the prior art.
Biological Tests
The following test procedures may be employed.
Test A
Determination of Thrombin Clotting Time (TT)
The inhibitor solution (25 xcexcL) is incubated with plasma (25 xcexcL) for three minutes. Human thrombin (T 6769; Sigma Chem. Co or Hematologic Technologies) in buffer solution, pH 7.4 (25 xcexcL, 4.0 NIH units/mL), is then added and the clotting time measured in an automatic device (KC 10; Amelung).
The thrombin clotting time (TT) is expressed as absolute values (seconds) as well as the ratio of TT without inhibitor (TT0) to TT with inhibitor (TTi). The latter ratios (range 1-0) are plotted against the concentration of inhibitor (log transformed) and fitted to sigmoidal dose-response curves according to the equation
y=a/[1+(x/IC50)S]
where: a=maximum range, i.e. 1; s=slope of the dose-response curve; and IC50=the concentration of inhibitor that doubles the clotting time. The calculations are processed on a PC using the software program GraFit Version 3, setting equation equal to: Start at 0, define end=1 (Erithacus Software, Robin Leatherbarrow, Imperial College of Science, London, UK).
Test B
Determination of Thrombin Inhibition with a Chromogenic, Robotic Assay
The thrombin inhibitor potency is measured with a chromogenic substrate method, in a Plato 3300 robotic microplate processor (Rosys AG, CH-8634 Hombrechtikon, Switzerland), using 96-well, half volume microtitre plates (Costar, Cambridge, Mass., USA; Cat No 3690). Stock solutions of test substance in DMSO (72 xcexcL), 0.1-1 mmol/L, are diluted serially 1:3 (24+48 xcexcL) with DMSO to obtain ten different concentrations, which are analysed as samples in the assay. 2 xcexcL of test sample is diluted with 124 xcexcL assay buffer, 12 xcexcL of chromogenic substrate solution (S-2366, Chromogenix, Mxc3x6lndal, Sweden) in assay buffer and finally 12 xcexcL of xcex1-thrombin solution (Human xcex1-thrombin, Sigma Chemical Co. or Hematologic Technologies) in assay buffer, are added, and the samples mixed. The final assay concentrations are: test substance 0.00068-13.3 xcexcmol/L, S-2366 0.30 mmol/L, xcex1-thrombin 0.020 NIHU/mL. The linear absorbance increment during 40 minutes incubation at 37xc2x0 C. is used for calculation of percentage inhibition for the test samples, as compared to blanks without inhibitor. The IC50-robotic value, corresponding to the inhibitor concentration which causes 50% inhibition of the thrombin activity, is calculated from a log concentration vs. % inhibition curve.
Test C
Determination of the Inhibition Constant Ki for Human Thrombin
Ki-determinations are made using a chromogenic substrate method, performed at 37xc2x0 C. on a Cobas Bio centrifugal analyser (Roche, Basel, Switzerland). Residual enzyme activity after incubation of human xcex1-thrombin with various concentrations of test compound is determined at three different substrate concentrations, and is measured as the change in optical absorbance at 405 nm.
Test compound solutions (100 xcexcL; normally in buffer or saline containing BSA 10 g/L) are mixed with 200 xcexcL of human xcex1-thrombin (Sigma Chemical Co) in assay buffer (0.05 mol/L Tris-HCl pH 7.4, ionic strength 0.15 adjusted with NaCl) containing BSA (10 g/L), and analysed as samples in the Cobas Bio. A 60 xcexcL sample, together with 20 xcexcL of water, is added to 320 xcexcL of the substrate S-2238 (Chromogenix AB, Mxc3x6lndal, Sweden) in assay buffer, and the absorbance change (xcex94A/min) is monitored. The final concentrations of S-2238 are 16, 24 and 50 xcexcmol/L and of thrombin 0.125 NIH U/mL.
The steady state reaction rate is used to construct Dixon plots, i.e. diagrams of inhibitor concentration vs. 1/(xcex94A/min). For reversible, competitive inhibitors, the data points for the different substrate concentrations typically form straight lines which intercept at x=xe2x88x92Ki.
Test D
Determination of Activated Partial Thromboplastin Time (APTT)
APTT is determined in pooled normal human citrated plasma with the reagent PTT Automated 5 manufactured by Stago. The inhibitors are added to the plasma (10 xcexcL inhibitor solution to 90 xcexcL plasma) and incubated with the APTT reagent for 3 minutes followed by the addition of 100 xcexcL of calcium chloride solution (0.025 M) and APTT is determined by use of the coagulation analyser KC10 (Amelung) according to the instructions of the reagent producer.
The clotting time is expressed as absolute values (seconds) as well as the ratio of APTT without inhibitor (APTT0) to APTT with inhibitor (APTTi). The latter ratios (range 1-0) are plotted against the concentration of inhibitor (log transformed) and fitted to sigmoidal dose-response curves according to the equation
y=a/[1+(x/IC50)S]
where: a=maximum range, i.e. 1; s=slope of the dose-response curve; and IC50=the concentration of inhibitor that doubles the clotting time. The calculations are processed on a PC using the software program GraFit Version 3, setting equation equal to: Start at 0, define end=1 (Erithacus Software, Robin Leatherbarrow, Imperial College of Science, London, UK).
IC50APTT is defined as the concentration of inhibitor in human plasma that doubled the Activated Partial Thromboplastin Time.
Test E
Determination of Thrombin Time ex vivo
The inhibition of thrombin after oral or parenteral administration of the compounds of the invention, dissolved in ethanol:Solutol(trademark):water (5:5:90), is examined in conscious rats which, one or two days prior to the experiment, are equipped with a catheter for blood sampling from the carotid artery. On the experimental day blood samples are withdrawn at fixed times after the administration of the compound into plastic tubes containing 1 part sodium citrate solution (0.13 mol per L) and 9 parts of blood. The tubes are centrifuged to obtain platelet poor plasma.
50 xcexcL of plasma samples are precipitated with 100 xcexcL of cold acetonitrile. The samples are centrifuged for 10 minutes at 4000 rpm. 75 xcexcL of the supernatant is diluted with 75 xcexcL of 0.2% formic acid. 10 xcexcL volumes of the resulting solutions are analysed by LC-MS/MS and the concentrations of thrombin inhibitor are determined using standard curves.
Test F
Determination of Plasma Clearance in Rat
Plasma clearance was estimated in male Sprague Dawley rats. The compound was dissolved in water and administered as a subcutaneous bolus injection at a dose of 4 xcexcmol/kg. Blood samples were collected at frequent intervals up to 5 hours after drug administration. Blood samples were centrifuged and plasma was separated from the blood cells and transferred to vials containing citrate (10% final concentration). 50 xcexcL of plasma samples are precipitated with 100 xcexcL of cold acetonitrile. The samples are centrifuged for 10 minutes at 4000 rpm. 75 xcexcL of the supernatant is diluted with 75 xcexcL of 0.2% formic acid. 10 xcexcL volumes of the resulting solutions are analysed by LC-MS/MS and the concentrations of thrombin inhibitor are determined using standard curves. The area under the plasma concentration-time profile was estimated using the log/linear trapezoidal rule and extrapolated to infinite time. Plasma clearance (CL) of the compound was then determined as
CL=Dose/AUC 
The values are reported in mL/min/kg.
Test G
Determination of in vitro Stability
Liver microsomes were prepared from Sprague-Dawley rats and human liver samples according to internal SOPs. The compounds were incubated at 37xc2x0 C. at a total microsome protein concentration of 3 mg/mL in a 0.05 mol/L TRIS buffer at pH 7.4, in the presence of the cofactors NADH (2.5 mmol/L) and NADPH (0.8 mmol/L). The initial concentration of compound was 5 or 10 xcexcmol/L. Samples were taken for analysis up to 60 minutes after the start of the incubation. The enzymatic activity in the collected sample was immediately stopped by adding 20% myristic acid at a volume corresponding to 3.3% of the total sample volume. The concentration of compound remaining (FINAL CONC) in the 60 min. sample was determined by means of LCMS using a sample collected at zero time as reference (START CONC). The % of degraded thrombin inhibitor was calculated as:   100  ⁢      xe2x80x83    ⁢  %  xc3x97                    [        STARTCONC        ]            -              [        FINALCONC        ]                    [      STARTCONC      ]      
Test H
Arterial Thrombosis Model
Vessel damage was induced by applying ferric chloride (FeCl3) topically to the carotid artery. Rats are anaesthetised with an intraperitoneal injection of sodium pentobarbital (80 mg/kg; Apoteksbolaget; Umexc3xa5, Sweden), followed by continuous infusion (12 mg/kg/h) throughout the experiment.
Rat body temperature was maintained at 38xc2x0 C. throughout the experiment by external heating. The experiment started with a 5 minutes control period. Five minutes later, human 125I-fibrinogen (80 kBq; IM53; Amersham International, Buckinghamshire, UK) was given intravenously and was used as a marker for the subsequent incorporation of fibrin(ogen) into the thrombus. The proximal end of the carotid artery segment was placed in a plastic tube (6 mm; Silastic(copyright); Dow Corning, Mich., USA) opened lengthways, containing FeCl3-soaked (2 xcexcL; 55% w/w; Merck, Darmstadt, Germany) filter paper (diameter 3 mm; IF; Munktell, Grycksbo, Sweden). The left carotid artery was exposed to FeCl3 for 10 minutes and was then removed from the plastic tube and soaked in saline. Fifty minutes later, the carotid artery was removed and rinsed in saline. Reference blood samples were also taken for determination of blood 125I-activity, 10 minutes after the injection of 125I-fibrinogen, and at the end of the experiment. The 125I-activity in the reference blood samples and the vessel segment were measured in a gamma counter (1282 Compugamma; LKB Wallac Oy, Turku, Finland) on the same day as the experiment was performed. The thrombus size was determined as the amount of 125I-activity incorporated in the vessel segment in relation to the 125I-activity in the blood (cpm/mg).
The invention is illustrated by way of the following examples.
General Experimental Details
TLC was performed on silica gel.
Chiral HPLC analysis was performed using a 46 mmxc3x97250 mm Chiralcel OD column with a 5 cm guard column. The column temperature was maintained at 35xc2x0 C. A flow rate of 1.0 mL/min was used. A Gilson 115 UV detector at 228 nm was used. The mobile phase consisted of hexanes, ethanol and trifluroacetic acid and the appropriate ratios are listed for each compound. Typically, the product was dissolved in a minimal amount of ethanol and this was diluted with the mobile phase.
LC-MS/MS was performed using a HP-1100 instrument equipped with a CTC-PAL injector and a 5 xcexcm, 4xc3x9710 mm ThermoQuest, Hypersil BDS-C18 column. An API-3000 (Sciex) MS detector was used. The flow rate was 1.2 mL/min and the mobile phase (gradient) consisted of 10-90% acetonitrile with 90-10% of 4 mM aq. ammonium acetate, both containing 0.2% formic acid.
1H NMR spectra were recorded using tetramethylsilane as the internal standard. 13C NMR spectra were recorded using the listed deuterated solvents as the internal standard.
Melting points are uncorrected.